The present invention relates generally to chemokines and more particularly to purified and isolated polynucleotides encoding a novel human C-C chemokine, to purified and isolated chemokine protein encoded by the polynucleotides, to chemokine analogs, to materials and methods for the recombinant production of the novel chemokine protein and analogs, to antibodies reactive with the novel chemokine, to chemokine inhibitors, and to uses of all of the foregoing materials.
Chemokines, also known as xe2x80x9cintercrinesxe2x80x9d and xe2x80x9cSIS cytokinesxe2x80x9d, comprise a family of small secreted proteins (e.g., 70-100 amino acids and about 8-10 kiloDaltons) which attract and activate leukocytes and thereby aid in the stimulation and regulation of the immune system. The name xe2x80x9cchemokinexe2x80x9d is derived from chemotactic cytokine, and refers to the ability of these proteins to stimulate chemotaxis of leukocytes. Indeed, chemokines may comprise the main attractants for inflammatory cells into pathological tissues. See generally, Baggiolini et al., Annu. Rev. Immunol, 15: 675-705 (1997); and Baggiolini et al., Advances in Immunology, 55:97-179 (1994), both of which are incorporated by reference herein. While leukocytes comprise a rich source of chemokines, several chemokines are expressed in a multitude of tissues. Baggiolini et al. (1994), Table II.
Previously identified chemokines generally exhibit 20-70% amino acid identity to each other and contain four highly-conserved cysteine residues. Based on the relative position of the first two of these cysteine residues, chemokines have been further classified into two subfamilies. In the xe2x80x9cC-X-Cxe2x80x9d or xe2x80x9cxcex1xe2x80x9d subfamily, encoded by genes localized to human chromosome 4, the first two cysteines are separated by one amino acid. In the xe2x80x9cC-Cxe2x80x9d or xe2x80x9cxcex2xe2x80x9d subfamily, encoded by genes on human chromosome 17, the first two cysteines are adjacent. X-ray crystallography and NMR studies of several chemokines have indicated that, in each family, the first and third cysteines form a first disulfide bridge, -and the second and fourth cysteines form a second disulfide bridge, strongly influencing the native conformation of the proteins. In humans alone, more than ten distinct sequences have been described for each chemokine subfamily. Chemokines of both subfamilies have characteristic leader sequences of twenty to twenty-five amino acids.
The C-X-C chemokines, which include IL-8, GROxcex1/xcex2/xcex3, platelet basic protein, Platelet Factor 4 (PF4), IP-10, NAP2, and others, share approximately 25% to 60% identity when any two amino acid sequences are compared (except for the GROxcex1/xcex2/xcex3 members, which are 84-88% identical with each other). Most of the C-X-C chemokines (excluding IP-10 and Platelet Factor 4) share a common E-L-R tri-peptide motif upstream of the first two cysteine residues, and are potent stimulants of neutrophils, causing rapid shape change, chemotaxis, respiratory bursts, and degranulation. These effects are mediated by seven-transmembrane-domain rhodopsin-like G protein-coupled receptors; a receptor specific for IL-8 has been cloned by Holmes et al., Science, 253: 1278-80 (1991), while a similar receptor (77% identity) which recognizes IL-8, GRO and NAP2 has been cloned by Murphy and Tiffany, Science, 253:1280-83 (1991). Progressive truncation of the N-terminal amino acid sequence of certain C-X-C chemokines, including IL-8, is associated with marked increases in activity.
The C-C chemokines, which include Macrophage Inflammatory Proteins MIP-1xcex1 and MIP-1xcex2, Monocyte chemoattractant proteins 1, 2, 3, and 4 (MCP-1/2/3/4), RANTES, 1-309, eotaxin, TARC, and others, share 25% to 70% amino acid identity with each other. Previously-identified C-C chemokines activate monocytes, causing calcium lux and chemotaxis. More selective effects are seen on lymphocytes, for example, T lymphocytes, which respond best to RANTES. Several seven-transmembrane-domain G protein-coupled receptors for C-C chemokines have been cloned to date, including a C-C chemokine receptor-1 (CCR1) which recognizes, e.g., MIP-1xcex1 and RANTES (Neote et al., Cell, 72:415-425 (1993)); a CCR2 receptor which has two splice variants and which recognizes, e.g., MCP-1 (Charo et al., Proc. Nat. Acad. Sci., 91:2752-56 (1994)); CCR3, which recognizes, e.g., eotaxin, RANTES, and MCP-3 (Combadiere, J. Biol. Chem., 270:16491 (1995)); CCR4, which recognizes MIP-1xcex1, RANTES, and MCP-1 (Power et al., J. Biol. Chem., 270:19495 (1995)); and CCR5, which recognizes MIP-1xcex1, MIP-1xcex2, and RANTES (Samson et al., Biochemstry, 35:3362 (1996)). Several CC chemokines have been shown to act as attractants for activated T lymphocytes. See Baggiolini et al. (1997).
Truncation of the N-terminal amino acid sequence of certain C-C chemokines also has been associated with alterations in activity. For example, mature RANTES (1-68) is processed by CD26 (a dipeptidyl aminopeptidase specific for the sequence NH2-X-Pro- . . . ) to generate a RANTES (3-68) form that is capable of interacting with and transducing a signal through CCR5 (like the RANTES (1-68) form), but is one hundred-fold reduced in its capacity to stimulate through the receptor CCR1. See Proost et al., J. Biol. Chem., 273(13): 7222-7227 (1998); and Oravecz et al., J. Exp. Med., 186: 1865-1872 (1997). U.S. Pat. No. 5,705,360 to Rollins and Zhang purports to describe N-terminal deletions of chemokine MCP-1 that inhibit receptor binding to the corresponding endogenous chemokine.
The roles of a number of chemokines, particularly IL-8, have been well documented in various pathological conditions. See generally Baggiolini et al. (1994), supra, Table VII. Psoriasis, for example, has been linked to over-production of IL-8, and several studies have observed high levels of IL-8 in the synovial fluid of inflamed joints of patients suffering from rheumatic diseases, osteoarthritis, and gout.
The role of C-C chemokines in pathological conditions also has been documented, albeit less comprehensively than the role of IL-8. For example, the concentration of MCP-1 is higher in the synovial fluid of patients suffering from rheumatoid arthritis than that of patients suffering from other arthritic diseases. The MCP-1 dependent influx of mononuclear phagocytes may be an important event in the development of idiopathic pulmonary fibrosis. The role of C-C chemokines in the recruitment of monocytes into atherosclerotic areas is currently of intense interest, with enhanced MCP-1 expression having been detected in macrophage-rich arterial wall areas but not in normal arterial tissue. Expression of MCP-1 in malignant cells has been shown to suppress the ability of such cells to form tumors in vivo. (See U.S. Pat. No. 5,179,078, incorporated herein by reference.) A need therefore exists for the identification and characterization of additional C-C chemokines, to further elucidate the role of this important family of molecules in pathological conditions, and to develop improved treatments for such conditions utilizing chemokine-derived products.
Chemokines of the C-C subfamily have been shown to possess utility in medical imaging, e.g., for imaging sites of infection, inflammation, and other sites having C-C chemokine receptor molecules. See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778, incorporated herein by reference. Such methods involve chemical attachment of a labeling agent (e.g., a radioactive isotope) to the C-C chemokine using art recognized techniques (see, e.g., U.S. Pat. Nos. 4,965,392 and 5,037,630, incorporated herein by reference), administration of the labeled chemokine to a subject in a pharmaceutically acceptable carrier, allowing the labeled chemokine to accumulate at a target site, and imaging the labeled chemokine in vivo at the target site. A need in the art exists for additional new C-C chemokines to increase the available arsenal of medical imaging tools.
The C-C chemokines RANTES, MIP-xcex1, and MIP-1xcex2 also have been shown to be the primary mediators of the suppressive effect of human T cells on the human immunodeficiency virus (HIV), the agent responsible for causing human Acquired Immune Deficiency Syndrome (AIDS). These chemokines show a dose-dependent ability to inhibit specific strains of HIV from infecting cultured T cell lines [Cocchi et al., Science, 270:1811 (1995)]. In addition, International patent publication number WO 97/44462, filed by Institut Pasteur, describes the use of fragments and analogs of the chemokine RANTES as antagonists, to block RANTES interaction with its receptors, for the purpose of suppressing HIV. The C-X-C chemokine stromal derived factor-1 (SDF-1) also is capable of blocking infection by T-tropic HIV-1 strains. See Winkler et al., Science, 279:389-393 (1998). However, the processes through which chemokines exert their protective effects have not been fully elucidated, and these chemokines in fact may stimulate HIV replication in cells exposed to the chemokines before HIV infection. See Kelly et al., J. Immunol., 160:3091-3095 (1998). Moreover, not all tested strains of the virus are equally susceptible to the inhibitory effects of chemokines; therefore, a need exists for additional C-C chemokines for use as inhibitors of strains of HIV.
Similarly, it has been established that certain chemokine receptors such as CCR5 [International Patent Publication No. WO 97/44055, published Nov. 27, 1997], CCR8, CCR2, and CXCR4) are essential co-receptors (with the CD4 receptor) for HIV-1 entry into susceptible cells, and that progression to AIDS is delayed in patients having certain variant alleles of these receptors. A need exists for additional therapeutics to inhibit HIV-1 infection and/or proliferation by interfering with HIV-1 entry and/or proliferation in susceptible cells.
More generally, due to the importance of chemokines as mediators of chemotaxis and inflammation, a need exists for the identification and isolation of new members of the chemokine family to facilitate modulation of inflammatory and immune responses.
For example, substances that promote inflammation may promote the healing of wounds or the speed of recovery from conditions such as pneumonia, where inflammation is important to eradication of infection. Modulation of inflammation is similarly important in pathological conditions manifested by inflammation. Crohn""s disease, manifested by chronic inflammation of all layers of the bowel, pain, and diarrhea, is one such pathological condition. The failure rate of drug therapy for Crohn""s disease is relatively high, and the disease is often recurrent even in patients receiving surgical intervention. The identification, isolation, and characterization of novel chemokines facilitates modulation of inflammation.
Similarly, substances that induce an immune response may promote palliation or healing of any number of pathological conditions. Due to the important role of leukocytes (e.g., neutrophils and monocytes) in cell-mediated immune responses, and due to the established role of chemokines in leukocyte chemotaxis, a need exists for the identification and isolation of new chemokines to facilitate modulation of immune responses.
Additionally, the established correlation between chemokine expression and inflammatory conditions and disease states provides diagnostic and prognostic indications for the use of chemokines, as well as for antibody substances that are specifically immunoreactive with chemokines; a need exists for the identification and isolation of new chemokines to facilitate such diagnostic and prognostic indications.
In addition to their ability to attract and activate leukocytes, some chemokines, such as IL-8, have been shown to be capable of affecting the proliferation of non-leukocytic cells. See Tuschil, J. Invest. Dermatol., 99:294-298 (1992). A need exists for the identification and isolation of new chemokines to facilitate modulation of such cell proliferation.
It will also be apparent from the foregoing discussion of chemokine activities that a need exists for modulators of chemokine activities, to inhibit the effects of endogenously-produced chemokines and/or to promote the activities of endogenously-produced or exogenously administered chemokines. Such modulators typically include small molecules, peptides, chemokine fragments and analogs, and/or antibody substances. Chemokine inhibitors interfere with chemokine signal transduction, i.e., by binding chemokine molecules, by competitively or non-competitively binding chemokine receptors, and/or by interfering with signal transduction downstream from the chemokine receptors. A need exists in the art for effective assays to rapidly screen putative chemokine modulators for modulating activity.
For all of the aforementioned reasons, a need exists for recombinant methods of production of newly discovered chemokines, which methods facilitate clinical applications involving the chemokines and chemokine inhibitors.
The present invention provides novel purified and isolated polynucleotides and polypeptides, antibodies, and methods and assays that fulfill one or more of the needs outlined above.
For example, the invention provides purified and isolated polynucleotides (i.e., DNA and RNA, both sense and antisense strands) encoding a novel human chemokine of the C-C subfamily, herein designated xe2x80x9cMacrophage Derived Chemokinexe2x80x9d or xe2x80x9cMDCxe2x80x9d. Preferred DNA sequences of the invention include genomic and cDNA sequences and chemically synthesized DNA sequences. Polynucleotides encoding non-human vertebrate forms of MDC, especially mammalian and avian forms of MDC, also are intended as aspects of the invention.
The nucleotide sequence of a cDNA, designated MDC cDNA, encoding this chemokine, is set forth in SEQ ID NO: 1, which sequence includes 5xe2x80x2 and 3xe2x80x2 non-coding sequences. A preferred DNA of the present invention comprises nucleotides 20 to 298 of SEQ ID NO. 1, which nucleotides comprise the MDC coding sequence.
The human MDC protein comprises a putative twenty-four amino acid signal sequence at its amino terminus. Another preferred DNA of the present invention comprises nucleotides 92 to 298 of SEQ ID NO. 1, which nucleotides comprise the putative coding sequence of the mature (secreted) MDC protein, without the signal sequence.
The amino acid sequence of human chemokine MDC is set forth in SEQ ID NO: 2. Preferred polynucleotides of the present invention include, in addition to those polynucleotides described above, polynucleotides that encode the amino acid sequence set forth in SEQ ID NO: 2, and that differ from the polynucleotides described in the preceding paragraphs only due to the well-known degeneracy of the genetic code.
Similarly, since twenty-four amino acids (positions xe2x88x9224 to xe2x88x921) of SEQ ID NO: 2 comprise a putative signal peptide that is cleaved to yield the mature MDC chemokine, preferred polynucleotides include those which encode amino acids 1 to 69 of SEQ ID NO: 2. Thus, a preferred polynucleotide is a purified polynucleotide encoding a polypeptide having an amino acid sequence comprising amino acids 1-69 of SEQ ID NO: 2.
Among the uses for the polynucleotides of the present invention is the use as a hybridization probe, to identify and isolate genomic DNA encoding human MDC, which gene is likely to have a three exon/two intron structure characteristic of C-C chemokines genes. (See Baggiolini et al. (1994), supra); to identify and isolate DNAs having sequences encoding non-human proteins homologous to MDC; to identify human and non-human chemokine genes having similarity to the MDC gene; and to identify those cells which express MDC and the conditions under which this protein is expressed. Polynucleotides encoding human MDC have been employed to successfully isolate polynucleotides encoding at least two exemplary non-human embodiments of MDC (rat and mouse). (See SEQ ID NOs: 35-38.)
Hybridization probes of the invention also have diagnostic utility, e.g., for screening for inflammation in human tissue, such as colon tissue. More particularly, hybridization studies using an MDC polynucleotide hybridization probe distinguished colon tissue of patients with Crohn""s disease (MDC hybridization detected in epithelium, lamina propria, Payer""s patches, and smooth muscle) from normal human colon tissue (no hybridization above background).
Generally speaking, a continuous portion of the MDC cDNA of the invention that is at least about 14 nucleotides, and preferably about 18 nucleotides, is useful as a hybridization probe of the invention. Thus, in one embodiment, the invention includes a DNA comprising a continuous portion of the nucleotide sequence of SEQ ID NO: 1 or of the non-coding strand complementary thereto, the continuous portion comprising at least 18 nucleotides, the DNA being capable of hybridizing under stringent conditions to a coding or non-coding strand of a human MDC gene. For diagnostic utilities, hybridization probes of the invention preferably show hybridization specificity for MDC gene sequences. Thus, in a preferred embodiment, hybridization probe DNAs of the invention fail to hybridize under the stringent conditions to other human chemokine genes (e.g., MCP-1 genes, MCP-2 genes, MCP-3 genes, RANTES genes, MIP-1xcex1 genes, MIP-1xcex2 genes, and 1-309 genes, etc.).
In another aspect, the invention provides a purified polynucleotide which hybridizes under stringent conditions to the non-coding strand of the DNA of SEQ ID NO: 1. Similarly, the invention provides a purified polynucleotide which, but for the redundancy of the genetic code, would hybridize under stringent conditions to the non-coding strand of the DNA of SEQ ID NO: 1. Exemplary stringent hybridization conditions are as follows: hybridization at 42xc2x0 C. in 5xc3x97SSC, 20 mM NaPO4, pH 6.8, 50% formamide; and washing at 42xc2x0 C. in 0.2xc3x97SSC. Those skilled in the art understand that it is desirable to vary these conditions empirically based on the length and the GC nucleotide base content of the sequences to by hybridized, and that formulas for determining such variation exist. [See, e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual. Second Edition, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1989).]
In another aspect, the invention includes plasmid and viral DNA vectors incorporating DNAs of the invention, including any of the DNAs described above or elsewhere herein. Preferred vectors include expression vectors in which the incorporated MDC-encoding cDNA is operatively linked to an endogenous or heterologous expression control sequence. Such expression vectors may further include polypeptide-encoding DNA sequences operably linked to the MDC-encoding DNA sequences, which vectors may be expressed to yield a fusion protein comprising the MDC polypeptide of interest.
In another aspect, the invention includes a prokaryotic or eukaryotic host cell stably transfected or transformed with a DNA or vector of the present invention. In preferred host cells, the mature MDC polypeptide encoded by the DNA or vector of the invention is expressed. The DNAs, vectors, and host cells of the present invention are useful, e.g., in methods for the recombinant production of large quantities of MDC polypeptides of the present invention. Such methods are themselves aspects of the invention. For example, the invention includes a method for producing MDC wherein a host cell of the invention is grown in a suitable nutrient medium and MDC protein is isolated from the cell or the medium.
Knowledge of DNA sequences encoding MDC makes possible determination of the chromosomal location of MDC coding sequences, as well as identification and isolation by DNA/DNA hybridization of genomic DNA sequences encoding the MDC expression control regulatory sequences such as promoters, operators, and the like.
According to another aspect of the invention, host cells may be modified by activating an endogenous MDC gene that is not normally expressed in the host cells or that is expressed at a lower level than is desired. Such host cells are modified (e.g., by homologous recombination) to express MDC by replacing, in whole or in part, the naturally-occurring MDC promoter with part or all of a heterologous promoter so that the host cells express MDC. In such host cells, the heterologous promoter DNA is operatively linked to the MDC coding sequences, i.e., controls transcription of the MDC coding sequences. See, for example, PCT International Publication No. WO 94/12650; PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955. The invention also contemplates that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multi-functional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydro-orotase) and/or intron DNA may be recombined along with the heterologous promoter DNA into the host cells. If linked to the MDC coding sequences, amplification of the marker DNA by standard selection methods results in co-amplification of the MDC coding sequences in such host cells.
The DNA sequence information provided by the present invention also makes possible the development, by homologous recombination or xe2x80x9cknockoutxe2x80x9d strategies [see, Capecchi, Science, 244: 1288-1292 (1989)], of rodents that fail to express functional MDC or that express a variant of MDC. Such rodents are useful as models for studying the activities of MDC, MDC variants, and MDC modulators in vivo. Rodents having a humanized immune system are useful as models for studying the activities of MDC and MDC modulators toward HIV infection and proliferation.
In yet another aspect, the invention includes purified and isolated MDC polypeptides. Mammalian and avian MDC polypeptides are specifically contemplated. A preferred peptide is a purified chemokine polypeptide having an amino acid sequence comprising amino acids 1 to 69 of SEQ ID NO: 2 (human mature MDC). Throughout the application, human mature MDC usually will be referred to simply as xe2x80x9cMDCxe2x80x9d or as xe2x80x9cmature MDCxe2x80x9d. In instances where context warrants, such as certain descriptions of experiments that involve both human and non-human mature MDCs and/or that involve MDC fragments and analogs, human mature MDC will sometimes be specifically referred to as xe2x80x9chumanxe2x80x9d and will sometimes be referred to as xe2x80x9cMDC (1-69).xe2x80x9d
Mouse and Rat MDC polypeptides of the invention are taught in SEQ ID NOs: 36 and 38. The sequence in SEQ ID NO: 36 depicts a complete murine MDC, consisting of a 24 residue leader peptide (residues xe2x88x9224 to xe2x88x921 of SEQ ID NO: 36) and a 68 residue murine mature MDC. The sequence in SEQ ID NO: 38 depicts a partial rat MDC, consisting of 13 residues of the leader peptide (residues xe2x88x9213 to xe2x88x921) and the complete 68 residue rat mature MDC.
The polypeptides of the present invention may be purified from natural sources, but are preferably produced by recombinant procedures, using the DNAs, vectors, and/or host cells of the present invention, or are chemically synthesized. Purified polypeptides of the invention may be glycosylated or non-glyclosylated, water soluble or insoluble, oxidized, reduced, etc., depending on the host cell selected, recombinant production method, isolation method, processing, storage buffer, and the like.
Moreover, an aspect of the invention includes MDC polypeptide analogs wherein one or more amino acid residues is added, deleted, or replaced from the MDC polypeptides of the present invention, which analogs retain one or more of the biological activities characteristic of the C-C chemokines, especially of MDC. The small size of MDC facilitates chemical synthesis of such polypeptide analogs, which may be screened for MDC biological activities (e.g., the ability to induce macrophage chemotaxis, or inhibit monocyte chemotaxis) using the many activity assays described herein. Alternatively, such polypeptide analogs may be produced recombinantly using well-known procedures, such as site-directed mutagenesis of MDC-encoding DNAs of the invention, followed by recombinant expression of the resultant DNAs.
In a related aspect, the invention includes polypeptide analogs wherein one or more amino acid residues is added, deleted, or replaced from the MDC polypeptides of the present invention, which analogs lack the biological activities of C-C chemokines or MDC, but which are capable of competitively or non-competitively inhibiting the binding of MDC polypeptides with a C-C chemokine receptor. Such polypeptides are useful, e.g., for modulating the biological activity of endogenous MDC in a host, as well as useful for medical imaging methods described above.
Certain specific analogs of MDC are contemplated to modulate the structure, intermolecular binding characteristics, and biological activities of MDC. For example, amino-terminal (N-terminal) and carboxy-terminal (C-terminal) deletion analogs (truncations) are specifically contemplated to change MDC structure and function. Among the amino terminal deletion analogs that are specifically contemplated are analogs wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino terminal residues have been deleted (i.e., deletions up to the conserved cysteine pair at positions 12 and 13 of human, murine, and rat mature MDC). As set forth in detail below, experimental data indicates that most or all of these analogs will possess reduced MDC biological activities and, in fact, will act as inhibitors of one or more biological activities of mature MDC.
Additionally, the following single-amino acid alterations (alone or in combination) are specifically contemplated: (1) substitution of a non-basic amino acid for the basic arginine and/or lysine amino acids at positions 24 and 27, respectively, of SEQ ID NO: 2; (2) substitution of a charged or polar amino acid (e.g., serine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine or cysteine) for the tyrosine amino acid at position 30 of SEQ ID NO: 2, the tryptophan amino acid at position 59 of SEQ ID NO: 2, and/or the valine amino acid at position 60 of SEQ ID NO: 2, and (3) substitution of a basic or small, non-charged amino acid (e.g., lysine, arginine, histidine, glycine, alanine) for the glutamic acid amino acid at position 50 of SEQ ID NO: 2. Specific analogs having these amino acid alterations are encompassed by the following formula (SEQ ID NO: 25):
wherein the amino acid at position 24 is selected from the group consisting of arginine, glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine, phenylalanine, tyrosine, tryptophan, aspartate, glutamate, asparagine, glutamine, cysteine, and methionine; wherein the amino acid at position 27 is independently selected from the group consisting of lysine, glycine, alanine, valine, leucine, isoleucine, proline, serine, threonine, phenylalanine, tyrosine, tryptophan, aspartate, glutamate, asparagine, glutamine, cysteine, and methionine; wherein the amino acid at position 30 is independently selected from the group consisting of tyrosine, serine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, and cysteine; wherein the amino acid at position 50 is independently selected from the group consisting of glutamic acid, lysine, arginine, histidine, glycine, and alanine; wherein the amino acid at position 59 is independently selected from the group consisting of tryptophan, serine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, and cysteine; and wherein the amino acid at position 60 is independently selected from the group consisting of valine, serine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, and cysteine. Such MDC polypeptide analogs are specifically contemplated to modulate the binding characteristics of MDC to chemokine receptors and/or other molecules (e.g., heparin, glycosaminoglycans, erythrocyte chemokine receptors) that are considered to be important in presenting MDC to its receptor. In one preferred embodiment, MDC polypeptide analogs of the invention comprise amino acids 1 to 69 of SEQ ID NO. 25.
The following additional analogs have been synthesized and also are intended as aspects of the invention: (a) a polypeptide comprising a sequence of amino acids identified by positions 1 to 70 of SEQ ID NO: 30; (b) a polypeptide comprising a sequence of amino acids identified by positions 9 to 69 of SEQ ID NO: 2; (c) a polypeptide comprising a sequence of amino acids identified by positions 1 to 69 of SEQ ID NO: 31; and (d) a polypeptide comprising a sequence of amino acids identified by positions 1 to 69 of SEQ ID NO: 32.
As set forth in detail below, experimental data indicates that the addition of as few as one additional amino acid at the amino terminus of human mature MDC is sufficient to confer useful MDC inhibitory properties to the resultant analog. Thus, all amino terminal addition analogs are contemplated as an aspect of the invention. Such addition analogs include the addition of one or a few randomly selected amino acids; the addition of common tag sequences (e.g., polyhistidine sequences, hemagglutinin sequences, or other sequences commonly used to facilitate purification); and chemical additions to the amino terminus (e.g., the addition of an amino terminal aminooxypentane moiety). See Proudfoot et al., J. Biol. Chem., 271:2599-2603 (1996); Simmons et al., Science, 276 (5310): 276-279 (1997).
Also as set forth in detail below, evidence exists that mature MDC is cleaved in vivo by a dipeptidyl amino peptidase, resulting in an MDC (3-69) form that exhibits at least some activities antagonistic to MDC. An additional aspect of the invention includes analogs wherein the proline at position 2 of a mature MDC (e.g., human, murine, and rat MDC) is deleted or changed to an amino acid other than proline. Such analogs are collectively referred to as xe2x80x9cMDCxcex94Pro2 polypeptides.xe2x80x9d Those MDCxcex94Pro2 polypeptides that retain MDC biological activities are contemplated as useful in all indications wherein mature MDC is useful; and are expected to be less susceptible to activity-destroying depeptidyl amino peptidases that recognize and cleave the sequence NH2-Xaa-Pro- (e.g., CD26). Those MDCxcex94Pro2 polypeptides that lack MDC biological activities are contemplated as being used as MDC inhibitors.
It will be appreciated that, while the foregoing analogs were often described with reference to human mature MDC, similar analogs of other vertebrate MDC""s, especially mammalian MDC""s, also are contemplated as aspects of the invention.
It also will be appreciated that it may be advantageous to express MDC or MDC analogs as fusions with immunoglobulin sequences, human serum albumin sequences, or other sequences, or to perform other standard chemical modifications, for the purpose of extending the serum half-life of the MDC or MDC analog. See, e.g., Yeh et al., Proc. Nat""l. Acad. Sci. U.S.A., 89(5): 1904-1908 (1992); Sambrook et al., supra. The definition of polypeptides of the invention is intended to encompass such modifications.
In related aspects, the invention provides purified and isolated polynucleotides encoding such MDC polypeptide analogs, which polynucleotides are useful for, e.g., recombinantly producing the MDC polypeptide analogs; plasmid and viral vectors incorporating such polynucleotides, and prokaryotic and eukaryotic host cells stably transformed with such DNAs or vectors.
In another aspect, the invention includes antibody substances (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric or humanized antibodies, antigen-binding fragments of antibodies, and the like) which are immunoreactive with MDC polypeptides and polypeptide analogs of the invention. Such antibodies are useful, e.g., for purifying polypeptides of the present invention, for quantitative measurement of endogenous MDC in a host, e.g., using well-known ELISA techniques, and for modulating binding of MDC to its receptor(s). The invention further includes hybridoma cell lines that produce antibody substances of the invention. Exemplary antibodies of the invention include monoclonal antibodies 252Y and 252Z, which are produced by hybridoma cell line 252Y and hybridoma cell line 252Z, respectively. The hybridoma cell lines are themselves aspects of the invention, and have been deposited with the American Type Culture Collection (ATCC Accession Nos. HB-12433 and HB-12434, respectively). Another exemplary antibody of the invention is monoclonal antibody 272D, which is produced by hybridoma cell line 272D (itself an aspect of the invention and deposited with the American Type Culture Collection (ATCC Accession No. HB-12498).
Recombinant MDC polypeptides and polypeptide analogs of the invention may be utilized in a like manner to antibodies in binding reactions, to identify cells expressing receptor(s) of MDC and in standard expression cloning techniques to isolate polynucleotides encoding the receptor(s). Such MDC polypeptides, MDC polypeptide analogs, and MDC receptor polypeptides are useful for modulation of MDC chemokine activity, and for identification of polypeptide and chemical (e.g., small molecule) MDC agonists and antagonists.
Additional aspects of the invention relate to pharmaceutical utilities of MDC polypeptides and polypeptide analogs of the invention. For example, MDC has been shown to modulate leukocyte chemotaxis. In particular, MDC has been shown to induce macrophage chemotaxis and to inhibit monocyte chemotaxis. Thus, in one aspect, the invention includes a method for modulating (e.g., up-regulating or down-regulating) leukocyte chemotaxis in a mammalian host comprising the step of administering to the mammalian host an MDC polypeptide or polypeptide analog of the invention, wherein the MDC polypeptide or MDC polypeptide analog modulates leukocyte chemotaxis in the host. In preferred methods, the leukocytes are monocytes and/or macrophages. For example, empirically determined quantities of MDC are administered (e.g., in a pharmaceutically acceptable carrier) to induce macrophage chemotaxis or to inhibit monocyte chemotaxis, whereas inhibitory MDC polypeptide analogs are employed to achieve the opposite effect.
In another aspect, the invention provides a method for palliating an inflammatory or other pathological condition in a patient, the condition characterized by at least one of (i) monocyte chemotaxis toward a site of inflammation in said patient or (ii) fibroblast cell proliferation, the method comprising the step of administering to the patient a therapeutically effective amount of MDC. In one embodiment, a therapeutically effective amount of MDC is an amount capable of inhibiting monocyte chemotaxis. In another embodiment, a therapeutically effective amount of MDC is an amount capable of inhibiting fibroblast cell proliferation. Such therapeutically effective amounts are empirically determined using art-recognized dose-response assays.
As an additional aspect, the invention provides a pharmaceutical composition comprising an MDC polypeptide or polypeptide analog of the invention in a pharmaceutically acceptable carrier. Similarly, the invention relates to the use of a composition according to the invention for the treatment of disease states, e.g., inflammatory disease states. In one embodiment, the inflammatory disease state is characterized by monocyte chemotaxis toward a site of inflammation in a patient having the disease state. In another embodiment, the disease state is characterized by fibroblast cell proliferation in a patient having the disease state.
MDC induced chemotaxis of natural killer cells (NK) can lead to enhanced cytotoxicity of targeted NK cells against carious forms of cancers. These forms of cancers include all solid tumor and cancerous cells found in various organs and skin (e.g., breast, ovarian, prostate, kidney, lung, pancreas, liver and bone cancers). NK cells also play an important role in antibody-dependent cell-mediated cytotoxicity. Stimulation of this process with MDC or MDC agonists would lead to improved immune response to tumors. [See generally Immunology (Ed. Kuby, J.) pp 304-6, W.H. Freeman and Co., New York, N.Y. (1992)]. Similarly, NK cells lead to viral immunity. MDC may be used to potentiate resistance to common viral diseases (e.g., influenza and rhinoviruses) by stimulating NK conferred viral immunity by stimulating antigen-specific TH memory cells. [Immunology Ed. Kuby J. pp 420-425, W.H. Freeman and Co. New York, N.Y. (1992)]. xe2x80x9cTreatmentxe2x80x9d as used herein includes both prophylactic and therapeutic treatment.
The apparent optimal concentration of mature MDC in receptor binding and chemotaxis experiments is about 10 ng/ml. Thus, for therapeutic methods involving the systemic administration of MDC (or MDC analogs retaining a desired MDC biological activity), doses and dosing schedules are preferably selected to maintain circulating concentrations in blood of about 0.1-10 ng/ml. Preferred approaches for preparing a dose and maintaining such levels in the bloods include administration of MDC in a bolus fashion, so as to administer approximately 0.1-10 mg of MDC. This administration is repeated in order to maintain the stated blood concentration. For example, MDC is stable at 1 mg/ml in phosphate-buffered saline (PBS) and is administered to experimental animals using this formulation. This formulation, either liquid or lyophilized and reconstituted, is suitable for human parenteral use, e.g., via intravenous injection. Other formulations can be devised to concentrate the protein drug and stabilize it for use years after its preparation. [See, e.g., Stability and Characterization of Protein and Peptide Drugs; Case Histories, Wang Y J and Pearlman R. (Eds.), Plenum Press, New York (1993) (describing methods for the preparation of cytokines and other similar protein drug formulations by the inclusion of a variety of excipients to maintain solubility and stability and minimize aggregation)]. Exemplary excipients include citrate, EDTA, detergents of the Tween family, zwittergent family, or pluronic family, and amino acids such as cysteine to maintain the proper oxido-reductant state.
In a second preferred approach, MDC is administered using any of a number of drug delivery methods that are known in the art to facilitate slow-release of the bioactive product. This can be accomplished as easily as employing intramusculature administration [see for example M. Groves in Parental Technology Manual, Second edition, M. J. Groves (Ed.), Interpharm Press, Inc., Prairie View, Ill., pp. 6-7 (1988)] to cause the MDC to be adsorbed into the blood stream over a delayed period of time. Alternatively, the MDC product can be delivered using a number of drug delivery methods [see for a general review L M Sanders, in Peptide and Protein Drug Delivery, V. H. L. Lee (Ed.), Marcel Dekker, Inc., New York, pp. 785-806 (1991)]. For example, MDC is incorporated into biodegradable microspheres, such as poly(lactic-co-glycolic acid of PLGA) microspheres as shown using Human Growth Hormone, [Tracy, Biotechnol. Progress, 14: 108-115 (1988)], or leuprolide acetate microspheres [Okada et al., Pharm. Res, 8: 787-791 (1991)] which can permit administrations as infrequently as once monthly. A variety of other drug delivery approaches will be apparent to those in the art, including dry powder formulations suitable for inhalation made available by Inhale Corporation, Palo Alto, Calif, and transdermal delivery made available by Alza Corporation, Palo Alto, Calif.
It will also be apparent from the teachings herein relating to the various activities of MDC that modulators of MDC activities, to inhibit the effects of endogenously-produced MDC and/or to promote the activities of endogenously-produced or exogenously administered MDC, have therapeutic utility. Such modulators typically include small molecules, peptides, chemokine fragments and analogs, and/or antibody substances. MDC inhibitors interfere with MDC signal transduction, e.g., by binding MDC molecules, by competitively or non-competitively binding MDC receptors on target cells, and/or by interfering with signal transduction in the target cells downstream from the chemokine receptors. Thus, in another aspect, the invention provides assays to screen putative chemokine modulators for modulating activity. Modulators identified by methods of the invention also are considered aspects of the invention.
In one embodiment, the invention provides a method for identifying a chemical compound having MDC modulating activity comprising the steps of: (a) providing first and second receptor compositions comprising MDC receptors; (b) providing a control composition comprising detectably-labeled MDC; (c) providing a test composition comprising detectably-labeled MDC and further comprising the chemical compound; (d) contacting the first receptor composition with the control composition under conditions wherein MDC is capable of binding to MDC receptors; (e) contacting the second receptor composition with the test composition under conditions wherein MDC is capable of binding to MDC receptors; (f) washing the first and second receptor compositions to remove detectably-labeled MDC that is unbound to MDC receptors; (g) measuring detectably-labeled MDC in the first and second receptor compositions; and (h) identifying a chemical compound having MDC modulating activity, wherein MDC modulating activity is correlated with a difference in detectably-labeled MDC between the first second receptor compositions.
As reported herein, the chemokine receptor CCR4 has been demonstrated to be a high affinity receptor for MDC. Thus, in a preferred embodiment of the foregoing method, the first and second receptor compositions comprise the MDC receptor that is CCR4. Since CCR4 is a membrane protein, a preferred embodiment for practicing the method is one wherein the first and second receptor compositions comprise CCR4-containing cell membranes derived from cells that express CCR4 on their surface. The cell membranes may be on intact cells, or may constitute an isolated fraction of cells that express CCR4. Cells that naturally express CCR4 and cells that have been transformed or transfected to express CCR4 recombinantly are contemplated.
In a related aspect, the invention provides a method for identifying a modulator of binding between MDC and CCR4, comprising the steps of: (a) contacting MDC and CCR4 both in the presence of, and in the absence of, a putative modulator compound; (b) detecting binding between MDC and CCR4; and (c) identifying a putative modulator compound in view of decreased or increased binding between MDC and CCR4 in the presence of the putative modulator, as compared to binding in the absence of the putative modulator. The contacting is performed, for example, by combining MDC with cell membranes that contain CCR4, in a buffered aqueous suspension.
In one embodiment, the method is performed with labeled MDC. In step (b), binding between MDC and CCR4 is detected by detecting labeled MDC bound to CCR4. In a preferred embodiment, the contacting step comprises contacting a suspension of cell membranes comprising CCR4 with a solution containing MDC. In a highly preferred embodiment, the method further comprises the steps of recovering the cell membranes from the suspension after the contacting step (e.g., via filtration of the suspension); and washing the cell membranes prior to the detecting step to remove unbound MDC.
In an alternative embodiment, the method is performed with a host cell expressing CCR4 on its surface. In step (b), binding between MDC and CCR4 is detected by measuring the conversion of GTP to GDP in the host cell.
In yet another alternative embodiment, the method is performed with a host cell that expresses CCR4 on its surface, and binding between MDC and CCR4 expressed in the host cell is detected by measuring cAMP levels in the host cell.
It will be appreciated that assays for modulators such as those described above are often performed by immobilizing (e.g., on a solid support) one of the binding partners (e.g., MDC or a fragment thereof that is capable of binding CCR4, or CCR4 or a fragment thereof that is capable of binding MDC). In a preferred variation, the non-immobilized binding partner is labeled with a detectable agent. The immobilized binding partner is contacted with the labeled binding partner in the presence and in the absence of a putative modulator compound capable of specifically reacting with MDC or CCR4; binding between the immobilized binding partner and the labeled binding partner is detected; and modulating compounds are identified as those compounds that affect binding between the immobilized binding partner and the labeled binding partner.
In yet another embodiment, the invention provides a method for identifying a chemical compound having MDC modulating activity, comprising the steps of: (a) providing first and second receptor compositions comprising MDC receptors; (b) contacting the first receptor composition with a control composition comprising detectably-labeled MDC; (c) contacting the second receptor composition with a test composition comprising detectably-labeled MDC and further comprising the chemical compound; (d) washing the first and second receptor compositions to remove detectably-labeled MDC that is unbound to MDC receptors; (e) measuring detectably-labeled MDC in the first and second receptor compositions after the washing; and (f) identifying a chemical compound having MDC modulating activity, wherein MDC modulating activity is correlated with a difference in detectably-labeled MDC between the first and the second receptor compositions.
In yet another embodiment, MDC binding to its receptor is measured by measurement of the activation of a reporter gene that has been coupled to the receptor using procedures that have been reported in the art for other receptors. See, e.g., Himmler et al., Journal of Receptor Research, 13:79-94 (1993).
MDC-binding fragments of high affinity receptors of MDC are specifically contemplated as inhibitor compounds of the invention; antibodies to such receptors also are contemplated as inhibitor compounds of the invention.
MDC""s involvement in various aspects of immune responses is described in detail below. Based on the involvement of MDC in immune response, the administration of MDC antagonists is indicated, for example, in the treatment anaphylaxis [Brown, A. F., J. Accid. Emerg. Med., 12(2):89-100 (1995)], shock [Brown (1995) supra], ischemia, reperfusion injury and central ischemia [Lindsberg et al., Ann. Neurol., 30(2):117-129 (1991)], atherogenesis [Handley et al., Drug Dev. Res., 7:361-375 (1986)], Crohn""s disease [Denizot et al., Digestive Diseases and Sciences, 37(3):432-437 (1992)], ischemic bowel necrosis/necrotizing enterocolitis [Denizot et al. (1992), supra, and Hsuch et al., Acta Pediat. Suppl., 396:11-17 (1994)], ulcerative colitis (Denziot et al. (1992), ischemic stroke [Satoh et al., Stroke, 23:1090-1092 (1992)], ischemic brain injury [Lindsberg et al., Stroke, 21:1452-1457 (1990) and Lindsberg et al. (1991), supra], systemic lupus erythematosus [Matsuzaki et al., Clinica Chimica Acta, 210:139-144 (1992)], acute pancreatitis [Kald et al., Pancreas, 8(4):440-442 (1993)], septicemia (Kald et al. (1993), supra), acute post-streptococcal glomerulonephritis [Mezzano et al., J. Am. Soc. Nephrol., 4:235-242 (1993)], pulmonary edema resulting from IL-2 therapy [Rabinovichi et al., J. Clin. Invest., 89:1669-1673 (1992)], ischemic renal failure [Grino et al., Annals of Internal Medicine, 121(5):345-347 (1994)]; pre-term labor [Hoffman et al., Am. J. Obstet. Gynecol., 162(2):525-528 (1990) and Maki et al., Proc. Natl. Acad. Sci. USA, 85:728-732 (1988)], adult respiratory distress syndrome [Rabinovichi et al., J. Appl. Phsiol., 74(4): 1791-1802 (1993); Matsumoto et al., Clin. Exp. Pharmocol. Physiol., 19:509-515 (1992); and Rodriguez-Roisin et al., J. Clin. Invest., 93:188-194 (1994)]. xe2x80x9cTreatmentxe2x80x9d as used herein includes both prophylactic and therapeutic treatment.
MDC acts as a chemoattractant for TH2 differentiated memory cells, which produce the cytokines IL-4, L-5, IL-10 and others. It is expected that, in some instances, MDC leads to an immune state in which TH1 cytokine driven responses are reduced. In such instances, antagonism of MDC would lead to a state in which TH1 cytokine driven responses are enhanced. Modulation of the TH1-TH2 balance may lead to enhanced xe2x80x9cimmune surveillance,xe2x80x9d and improved eradication of viral and parasitic infections. Administration of MDC antagonists of the invention to mammalian subjects, especially humans, for the purposes of ameliorating pathological conditions associated with undesirable or excessive TH2 responses and/or less-than-desirable TH1 responses are contemplated as additional aspects of the invention. Administration of sufficient MDC antagonists to substantially reduce endogenous IL-10, a TH1 immune suppressing cytokine, would lead to enhanced cytotoxic T-lymphocyte mediated immunity and immune surveillance [see Muller et al., J. Infect. Dis., 177: 586-94 (1998); Kenney et al., J. Infect. Dis., 177: 815-9 (1998)]. In these situations an effective dose and dosing schedule can be determined by monitoring circulating IL-10 levels and increasing the dose and frequency of administration to reduce IL-10 levels to near normal levels. Treatment of chronic or persistent viral infections and parasitic infections is specifically contemplated, especially in combination with other antiviral or anti-parasitic infection therapeutics. Similarly, treatment or prevention of graft failure or graft rejection with MDC antagonists is contemplated. The administration of MDC antagonists is indicated, for example, in Leishmaniasis [Li et al., Infect. Immunol., 64:5248-5254 (1996); Krishnan et al., J. Immunol., 156(2):653-62 (1996)], opportunistic lung infections in cystic fibrosis patients [Moser et al., APMIS, 105(11):838-42 (1997)], to delay HIV-1 induced immunodeficiency [Berger et al., Res. Virol., 147(2-3):103-108 (1996); Barker et al., Proc. Natl. Acad. Sci. USA, 92(24): 11135-9 (1995); Jason et al., J. Acquir. Immune. Defic. Syndrome Retrovirol., 10(4): 471-6 (1995); Maggi et al., J. Biol. Regul. Homeost. Agents, 9(3): 78-81 (1995)], chronic interstitial lung disease [Kunkel et al., Sarcoidosis Vasc. Diffuse Lung Dis., 13: 120-128 (1996)], in neurological disorders associated with a TH2 response [Windhagen et al., Chem. Immunol., 63: 171-86 (1996); Bai et al., Clin. Immunol. Immunopathol., 83(2): 117-26 (1997)], colorectal cancer [Pellegrini et al., Cancer Immunol. Immunother., 42(1): 1-8 (1996)], viral infection, for example various species of herpes and hepatitis [Spruance et al., Antiviral Res., 28(1): 39-55 (1995); Pope et al., J. Immunol., 56(9): 3342-9 (1996); Bartoletti et al., Gastroenterol., 112(1): 193-199 (1997)], candidiasis and other fungal infections [Spaccapelo et al., J. Immunol., 155(3): 1349-60 (1995); Fidel et al., J. Infect. Dis., 176(3): 728-39 (1995); Cenci et al., J. Infect Dis., 171(5): 1279-88 (1995)], chronic pneumonia [Johansen et al., Behring Inst. Mitt., 98: 269-73 (1997)], solid tumor cancer [Khar et al., Cytokines Mol. Ther., 2(1): 39-46 (1996)], Bordella pertussis respiratory infection [Ryan et al., J. Infect Dis., 175(5): 1246-50 (1997)], systemic lupus erythrematosus [Nakamura et al., J. Immunol., 158(6): 2648-53 (1997)], Bullous pemphigoidd pathogenesis [Deptia et al., Arch. Dermatol Res., 289(12): 667-70 (1997)], glomerulonephritis [Kitching et al., Kidney Int., 53(1): 112-8(1998); Huang et al., J. Am Soc. Neprol., 8(7): 1101-8 (1997); Carballido et al., Eur. J. Immunol., 27(2): 515-21 (1997)], pulmonary respiratory syncytial virus infection [Hussell e al., Eur. J. Immunol., 27(12): 3341-9 (1997)], complications of trauma associated with surgical stress [Decker et al., Surgery, 119(3): 316-25 (1996)], celiac disease [Karban et al., Isr. J. Med. Sci., 33(3): 209-14 (1997)], Gulf War syndrome [Rook et al., Lancet, 349(9068): 1831-3 (1997)], ameobocyte infection, for example Plasmodium falciparum [Elghazali et al., Clin. Exp. Immunol., 109(1): 84-9 (1997)] and schistosoma mansoni [Wolowczuk et al., Immunol., 91(1): 35-44 (1997)], and B-cell lymphoma, especially mucosa-associated lymphoid tissue type [Greiner et al., Am J. Pathol., 150(5): 1583-93 (1997)]. xe2x80x9cTreatmentxe2x80x9d as used herein includes both prophylactic and therapeutic treatment.
With respect to any of the conditions, disorders, and disease states identified in the preceding paragraphs, an exemplary method of treatment comprises the steps of identifying a human subject in need of therapeutic or prophylactic treatment for one of the above-identified conditions, disorders, or disease states; and administering to the human subject a therapeutically or prophylactically effective amount of an MDC antagonist compound. By xe2x80x9ctherapeutically effective amountxe2x80x9d is meant a dose and dosing schedule that is sufficient to cure the disease state, or to reduce the symptoms or severity of the disease state. By xe2x80x9cprophylactically effective amountxe2x80x9d is meant a dose and dosing schedule that is sufficient to reduce the likelihood of occurrence of a disease state, or delay its onset, relative to human subjects that are considered to have equivalent risk of developing the disease state but whom are not treated with an MDC antagonist. Therapeutically effective amounts are readily determined by dose-response studies that are conventionally performed in the art.
In one highly preferred embodiment, the invention includes a method of inhibiting proliferation of a mammalian immunodeficiency virus comprising the step of contacting mammalian cells that are infected with a mammalian immunodeficiency virus with a composition comprising an MDC-IV antagonist compound or TARC-IV antagonist compound, in an amount effective to inhibit proliferation of said virus in said cells. The family of mammalian immunodeficiency viruses is intended to include human immunodeficiency viruses, such as strains of HIV-1 and HIV-2, and analogous viruses known to infect other mammalian species, including but not limited to simian and feline immunodeficiency viruses. The method can be performed in vitro (e.g., in cell culture), but preferably is performed in vivo by administering the antagonist to an infected subject, e.g., an HIV-infected human subject. (In yet another embodiment, the method is performed prophylacticly on a subject at risk of developing an HIV infection, e.g., due to the subject""s likelihood of exposure to contaminated blood samples, contaminated needles, or intimate exposure to an HIV-infected person.)
The term xe2x80x9cMDC-IV antagonist compoundxe2x80x9d refers to compounds that antagonize the apparent Immunodeficiency Virus-proliferative effects of MDC in infected cells. Thus, the term xe2x80x9cMDC-IV antagonist compoundxe2x80x9d is meant to include any compound that is capable of inhibiting proliferation of the immunodeficiency virus in a manner analogous to either the inhibition reported herein for MDC neutralizing antibodies or the inhibition reported herein for certain MDC analogs (e.g., analogs having amino terminal additions or truncations). For example, anti-MDC antibodies are highly preferred MDC-IV antagonist compounds. For treatment of humans infected with an HIV virus, humanized antibodies are highly preferred. Similarly, polypeptides that comprise an antigen-binding fragment of an anti-MDC antibody and that are capable of binding to MDC are preferred MDC-IV antagonist compounds.
As described elsewhere herein in greater detail, amino-terminal truncations of mature human MDC (1-69) possess antiproliferative activity against HIV-1. Thus, another set of preferred MDC-IV antagonist compounds are polypeptides whose amino acid sequence consists of a portion of the amino acid sequence set forth in SEQ ID NO: 2 sufficient to bind to the chemokine receptor CCR4, said portion having an amino-terminus between residues 3 and 12 of SEQ ID NO: 2 (i.e., analogs lacking at least three amino acids from the amino terminus of MDC (1-69). Amino terminal deletion analogs that have been further modified, e.g., by including an oligopeptide tag to facilitate purification, or by including an initiator methionine for bacterial expression, are also contemplated.
Amino-terminal additions to mature MDC also result in analogs possessing antiproliferative activity against HIV-1. Thus, another set of preferred MDC-IV antagonist compounds are polypeptides that comprise a mature MDC sequence (e.g., amino acids 1-69 of SEQ ID NO: 1), and that further comprise a chemical addition to the amino terminus of the mature MDC sequence to render said polypeptide antagonistic to MDC. Additions of additional amino acids and other chemical moieties are contemplated.
It will further be appreciated that substitution of amino acids in a mature MDC sequence (especially substitutions in the amino terminus of mature MDC) may be expected, in some instances, to result in analogs possessing antiproliferative activity against HIV-1. Such analogs also are intended MDC-IV antagonist compounds, and are identifiable using HIV proliferation assays described herein.
It is postulated that MDC""s HIV-proliferative effects are mediated, at least in part, through the chemokine receptor CCR4. Thus, the family of MDC-IV antagonist compounds includes polypeptides that comprise the C-C chemokine receptor 4 (CCR4) amino acid sequence set forth in SEQ ID NO: 34 or that comprise a continuous fragment thereof that is capable of binding to MDC or TARC. Such polypeptides are expected to bind endogenous MDC and thereby inhibit HIV proliferation in a manner analogous to anti-MDC antibodies. Also contemplated are anti-CCR4 antibodies, which are expected to block MDC-CCR4 interactions, thereby inhibiting MDC-induced HIV proliferation.
As described herein in detail, the chemokine TARC possesses sequence similarity to MDC, possesses various overlapping biological activities, and, like MDC, binds to the chemokine receptor CCR4. These similarities suggest that compounds that inhibit TARC-CCR4 interactions will also be useful for inhibiting proliferation of immunodeficiency viruses. Compounds that inhibit TARC-induced proliferation of such viruses are collectively referred to as xe2x80x9cTARC-IV antagonist compounds.xe2x80x9d Such compounds include anti-CCR4 antibodies, anti-TARC antibodies (especially humanized versions), and polypeptides that are capable of binding to TARC and that comprise an antigen-binding fragment of an anti-TARC antibody.
It is also contemplated that modifications to the amino terminus of mature TARC polypeptides will result in TARC-IV antagonist compounds, in a manner analogous to what has been reported herein for MDC analogs. Thus, TARC-1 V antagonist compounds for use in methods of the invention include polypeptides that have an amino acid sequence consisting of a portion of the amino acid sequence set forth in SEQ ID NO: 43 that is sufficient to bind to the chemokine receptor CCR4, said portion having an amino-terminus between residues 1 and 10 of SEQ ID NO: 43. Polypeptide comprising mature TARC sequences, and further comprising chemical additions to the amino terminus to render the polypeptide antagonistic to TARC also are contemplated. Polypeptides comprising the mature TARC amino acid sequence, into which substitutions have been introduced to confer HIV antiproliferative activity, also are contemplated as TARC-IV antagonist compounds.
In another highly preferred embodiment, the invention includes a method of inhibiting platelet aggregation in a mammalian subject (especially a human subject) comprising the step of administering to a mammalian subject a composition comprising an MDC-PA antagonist compound or TARC-PA antagonist compound, in an amount effective to inhibit platelet aggregation in the subject. Such methods may be performed for therapeutic purposes, e.g., in patients suffering from undesirable blood clotting, or for prophylactic purposes on a subject at risk of developing undesirable blood clotting or coagulation. Such patients would include, e.g., patients who have previously suffered myocardial infarction or stroke or other clotting disorders, or who are deemed to be at high risk for developing such conditions.
The term xe2x80x9cNDC-PA antagonist compoundxe2x80x9d refers to compounds that antagonize the apparent Platelet Aggregating effects of MDC. Thus, the term xe2x80x9cMDC-PA antagonist compoundxe2x80x9d is meant to include any compound that is capable of inhibiting platelet aggregation that is observable after administration of MDC to a mammalian subject (e.g., to a mouse or rat). Those compounds described above as MDC-IV antagonist compounds are specifically contemplated as MDC-PA antagonist compounds as well. For example, anti-MDC antibodies are highly preferred MDC-PA antagonist compounds. For treatment of humans, humanized antibodies are highly preferred. Similarly, polypeptides that comprise an antigen-binding fragment of an anti-MDC antibody and that are capable of binding to MDC are preferred MDC-PA antagonist compounds. All MDC analogs that inhibit the platelet aggregating effects of MDC also are preferred. Analogs having additions, deletions, and/or substitutions in the amino terminus are specifically contemplated.
The structural and functional similarities between MDC and TARC reported herein indicate that compounds that inhibit TARC-CCR4 interactions will be useful for inhibiting platelet aggregation. Compounds that inhibit TARC-induced platelet aggregation are collectively referred to as xe2x80x9cTARC-PA antagonist compounds.xe2x80x9d Such compounds include anti-CCR4 antibodies, anti-TARC antibodies (especially humanized versions); various TARC analogs described elsewhere herein, and polypeptides that are capable of binding to TARC and that comprise an antigen-binding fragment of an anti-TARC antibody.
As described herein in detail, the expression patterns of MDC and its receptor, CCR4, provide an indication for the use of MDC as an adjuvant in a vaccine. Thus, in another aspect, the invention includes a vaccine composition comprising an antigen of interest in a suitable pharmaceutical carrier, improved by the inclusion of MDC in the vaccine composition. The antigen of interest may be any composition intended to generate a desirable immune response in a human or other animal. Such compositions would include, for example, killed or attenuated pathogens or antigenic portions thereof In a related aspect, the invention includes a method of immunizing a human or animal, wherein the improvement comprises administering MDC to the human or animal, either concurrently or before or after administering an antigen of interest.
The foregoing aspects and numerous additional aspects will be apparent from the drawing and detailed description which follow.